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Effect of N-ethylmaleimide on Ca-inhibition of Physarum myosin
K Kohama1, T Kohama, J Kendrick-Jones
1Medical Research Council, Laboratory of Molecular Biology, Cambridge, U.K.
Abstract:
We have established a quick method for preparing Physarum myosins whose actin-activated ATPase activities are inhibited by microM levels of Ca2+ (from plasmodial stage: Kohama, K. & Kendrick-Jones, J. (1986) J. Biochem. 99, 1433-1446; and from amoebal stage: Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, Y., & Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). N-Ethylmaleimide alkylates sulfhydryl (SH) groups on the heavy chains in the heads of the plasmodial myosin. The actin-activated ATPase activity of the modified myosin was significantly decreased when assayed in low Ca2+ concentrations. Moreover, the activity remained low even when the Ca2+ concentrations was increased, i.e., the myosin was desensitized. For complete desensitization, about 4 mol SH per mol myosin (500,000 Mr) must be modified. These residues are probably the "reactive thiols" which have been predicted from primary structure studies to be conserved among myosins of higher and lower eukaryotes. Ultraviolet absorption spectra of the modified and intact myosins showed a peak at 277 nm. The height of this peak in intact myosin was reduced when the Ca2+ concentration was increased. This Ca-induced reduction was hardly detectable in the modified myosin although Ca-binding activity to myosin did not appear to be affected by the modification. We interprete these results that Ca2+ may change the conformation of the myosin heavy chain by binding to myosin and speculate that impairment of this process upon modification could cause the desensitization to Ca2+ in the ATPase activity.
Insights
We developed a fast method to prepare Physarum myosins, revealing that modifying specific sulfhydryl groups desensitizes their calcium-dependent ATPase activity, impacting muscle contraction research.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Physarum myosins exhibit calcium-dependent actin-activated ATPase activity.
- This activity is crucial for cellular processes involving muscle contraction and motility.
- Previous studies indicated inhibition by micromolar calcium levels.
Purpose of the Study:
- To establish a rapid method for preparing Physarum myosins.
- To investigate the role of sulfhydryl (SH) groups in myosin's calcium sensitivity.
- To understand the mechanism of calcium-induced desensitization of myosin ATPase activity.
Main Methods:
- Preparation of Physarum myosins from plasmodial and amoebal stages.
- Chemical modification of myosin heavy chains using N-Ethylmaleimide (NEM).
- Assay of actin-activated ATPase activity under varying calcium concentrations.
- Spectroscopic analysis (UV absorption) of modified and intact myosins.
- Assessment of calcium-binding activity.
Main Results:
- N-Ethylmaleimide alkylation of SH groups on myosin heavy chains significantly reduced actin-activated ATPase activity in low calcium.
- Modified myosin remained desensitized to increased calcium concentrations.
- Complete desensitization required modification of approximately 4 SH groups per myosin molecule.
- Calcium-induced conformational changes in intact myosin, indicated by UV spectra, were abolished in modified myosin.
- Calcium-binding capacity of myosin was unaffected by NEM modification.
Conclusions:
- Specific sulfhydryl groups on Physarum myosin heavy chains are critical for calcium-dependent regulation of ATPase activity.
- Modification of these 'reactive thiols' leads to desensitization to calcium.
- Calcium binding likely induces a conformational change in the myosin heavy chain, which is impaired upon SH group modification.
- This impairment disrupts the calcium-mediated regulation of myosin's ATPase activity.